S160: GATA2B HAPLOINSUFFICIENCY CAUSES ABERRANT TRANSCRIPTIONAL REGULATORY SIGNATURES IN HSPCS RESULTING IN MYELOID AND ERYTHROID DYSPLASIA IN ZEBRAFISH
Authors: Wei Zhang, Emanuele Gioacchino, Cansu Koyunlar, Joke Zink, Hans de Looper, Kirsten J. Gussinklo, Remco Hoogenboezem, Dennis Bosch, Eric Bindels, Ivo P. Touw, Emma de Pater
Journal: HemaSphere (2023)

Topic: 9. Myelodysplastic syndromes - Biology & Translational Research Background: The transcription factor GATA2 has pivotal roles in hematopoiesis. Germline GATA2 mutations in patients result in GATA2 haploinsufficiency syndrome characterized by immunodeficiency, bone marrow failure, and predispositions to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). It is unclear how GATA2 haploinsufficiency drives abnormal hematopoiesis, and to date, these phenotypes have not been captured in a cell or animal model to study the mechanism behind GATA2 haploinsufficiency induced bone marrow failure and leukemia predisposition. Aims: Our aim is to establish a model for GATA2 haploinsufficiency syndrome and to study the molecular mechanism driving leukemia predisposition. Methods:gata2b heterozygous (gata2b+/-) zebrafish were generated by CRISPR/Cas9 targeting exon 3. Adult kidney marrow (KM, bone marrow equivalent) was analysed by morphology, flow cytometry and single-cell (sc) RNA and ATAC sequencing. Results: Morphological analysis and cell counting showed that wild type (WT) KM cells had normal morphology, while gata2b+/- KM samples showed considerable erythroid and myeloid dysplasia (Fig A, a-b). scATAC-seq data suggested the chromatin accessibility of gata2b was increased in gata2b+/- HSPCs, including gene body region and upstream (+3.5-4.1kb and +5.4-6.2kb) regions (Fig C). Co-accessibility analysis suggested these two upstream regions might be enhancers for gata2b. Furthermore, we found that the co-accessibility between transcription start site (TSS) and +3.5-4.1kb enhancers was stronger in gata2b+/- zebrafish. This is suggestive of an auto-regulatory feedback mechanism, where Gata2 expression remains at sufficient levels after the loss of a single allele, underscored by no significant changes in gata2b expression levels in HSCs as marked by CD41:GFPlow expression. Because of this feedback mechanism, gata2b+/- chromatin is more accessible in the myeloid lineage. This causes a plethora of defects. scRNA-seq data revealed that erythroid dysplasia in gata2b+/- zebrafish was preceded by a differentiation block in erythroid progenitors, hallmarked by downregulation of cytoskeletal transcripts, aberrant proliferative signatures and ribosome biogenesis (Fig D, a). Cell-cell communication showed a reduction of the incoming strength of communication signals in the myeloid progenitor cells (Fig D, b) indicating a decreased reception ability of these cells. This was likely caused by the decreased expression of cd34 in gata2b+/- zebrafish progenitor cells. This weakened the adhesion of HSPCs with other progenitor cells (Fig D, c). Summary/Conclusion: Here, we create a model for GATA2 haploinsufficiency syndrome by generating heterozygous gata2b zebrafish showing abnormal hematopoiesis, characterized by myeloid and erythroid dysplasia. We observe gata2b chromatin over-accessibility in gata2b+/- zebrafish, and find that this resulted in reduced cell-cell interaction likely causing the defect in differentiation. (Wei Zhang and Emanuele Gioacchino contributed equally) Keywords: Zebra fish, HSC, GATA-2, Myelodysplastic syndrome